This invention relates to immunohematology procedures, and more particularly to a reagent system and a method for the identification of classes as well as subclasses within a class and enumeration of cells within those subclasses of blood leukocytes from a whole blood sample which has been incubated with a fluorescent responsive antibody to a select antigenic determinant on the surface of specified subclasses of blood leukocytes.
It is known that the lymphocyte population of blood leukocytes is subdivided into a number of subclasses which play distinct roles in the immune response. In disease states the relative number of lymphocytes found in various subclasses is likely to change. Hence, the enumeration and identification of the cells in the various subclasses will provide useful information in the study and treatment of disease as described by James R. Downing et al in Laboratory Management, May 1984, pages 29-37.
It is known that several particular subclasses of functionally distinct lymphocytes and other blood leukocytes can be identified on the basis of antigenic determinants on the cell.
Monoclonal antibody techniques have been utilized to produce large quantities of highly purified antibody to various lymphocyte and other leukocyte subclasses. Utilizing such antibodies, it has proved feasible to assay the lymphocytes of an individual to determine the relative number of cells in various subclasses. Further, utilizing direct or indirect techniques, the antibodies can be labeled fluorescently, thereby rendering the samples under consideration amenable to flow cytometric analysis and morphology. More recently, additional monoclonal antibodies have been developed which include several that react with monocytes and granulocytes.
Hansen et al described in U.S. Pat. No. 4,284,412, 1981, and in Immunology, Vol. 77, No. 8, pp 4914-4917 (1980) a method and apparatus for automated identification an enumeration of specified lymphocyte subclasses. An anticoagulated whole blood sample or buffy coat sample is incubated with an antibody to a specific lymphocyte subclass of interest. The binding of this antibody is detectable if either it has been coupled with a fluorescent chemical moiety (the direct technique), or if it in turn is specifically bound by another macromolecule to which has been coupled a fluorescent dye moiety (the indirect technique). These fluorescent moieties possess the characteristic of emitting fluorescent light upon illumination with incident laser light. The sample then is lysed using ammonium chloride as the lysing agent. A diluted sample then is subjected to flow cytofluorometric analysis. Four clusters of cells are distinguished. However, only three clusters are found to be due to leukocytes. These clusters were identified as (1) lymphocytes, (2) monocytes and (3) granulocytes. The fourth cluster is identified as aggregates or multiples of platelets and red blood cell debris due to incomplete lysing of the red blood cells.
The lysing techniques described in these references now have been improved by the present invention so as to maintain better the morphology of the immunologically labeled specific leukocytes, improve their stability on storage, and render them more suitable for cytofluorescent analysis, or for other operations such as microscopic examination of stained cells on a slide.
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